Using recombinant DNA methods we propose to elucidate the genetic mechanisms which control the expression of multiple forms of cytochrome P-450, which have different but overlapping substrate specificities. We will isolate cDNA clones for the phenobarbital, methylcholanthrene and isosafrole induced forms at rat liver cytochrome P-450 and determine the sequences of the cDNA inserts and of the proteins encoded by the respective mRNAs. These cDNAs will be used as heterologous probes to isolate cloned cDNA's for other expressed forms of cytochrome P-450. They will also be used as probes to isolate the corresponding genes, as well as related genes from a rat liver-Charon 4A genomic DNA library. We will compare the structures of the various specific mRNAs and genes and the arrangement of introns and exons within the different genes by biochemical and electron microscopic methods. This may lead to the identification of homologous domains within different molecules, which determine common functions, such as heme binding and the disposition of the polypeptide within the membrane. The separation of the coding regions in different exons may be correlated with the existence of distinct structural or functional domains within the P-450 proteins. The data will be assessed in terms of evolutionary relationships between members of a multigene family and the possibility that differential processing of the product of one or more transcription units plays an important role in generating P-450 diversity. We will also investigate the mechanism of cytochrome P-450 induction by various specific agents with respect to the possible involvement of transcriptional and posttranscriptional processes in the enhancement of cytoplasmic levels of specific mRNA species. DNA sequences involved in the regulation of P-450 gene transcription will be identified by constructing chimeric genes bearing putative regulatory elements and obtaining expression of these genes after transfection into hepatoma cells. The effect of various deletions or specific mutations, effected in vitro, on the inducibility of the exogenous genes will be assessed. If possible, we will construct chimeric genes coding for hybrid forms of cytochrome P-450. The chimeric proteins produced by expression of these genes will be analyzed for substrate specificity in order to identify the region(s) of each polypeptide involved in substrate recognition.